This proposal builds upon our recent findings that treatment with a clinically available antibiotic, Azithromycin (AZ), reduces brain damage and improves functional outcomes in a neonatal rodent hypoxic-ischemic (HI) brain injury model. Our aims are to refine and extend these results, build the foundation for testing AZ in larger animal HI models, and ultimately, repurpose AZ for treatment of neonates with hypoxic-ischemic encephalopathy (HIE). Neonatal HIE remains an important cause of disability. Although wide implementation of therapeutic hypothermia in US newborn intensive care units (NICUs) has improved outcomes of HIE, many survivors develop chronic neurological disorders; thus supplemental neuroprotection interventions are greatly needed. Moreover, in low resource settings with limited access to NICU care, therapeutic hypothermia may worsen outcomes. Thus, safe and effective treatments are urgently needed for neonates at risk for HIE- associated brain injury, both in US NICUs and in settings with no current effective treatment options. Our rationale for initial evaluation of AZ stemmed from: i) interest in understanding the roles of inflammation in neonatal HI injury and recovery, coupled with evidence that AZ modulates myeloid function; ii) a recent report that AZ confers protection in a murine adult stroke model; iii) knowledge that AZ is increasingly used intra- partum in obstetric practice with many newborns exposed to AZ (by trans-placental transport) without evidence of adverse effects; iv) reports that trials of AZ for treatment of neonatal infections identified no safety concerns. Based on these factors and our initial findings that AZ regulates microglia and stimulates their healing responses in neonatal brain, we evaluated the impact of AZ treatment on the outcome of HI brain injury in neonatal rodents. We used a well-characterized model of neonatal HI brain injury elicited in 7 day old rats by unilateral carotid artery ligation and timed exposure to a reduced (8%) oxygen environment; this HI model results in quantifiable sensorimotor deficits and unilateral forebrain damage. This model has been used for over 35 years to study mechanisms of neonatal HI brain injury and to test potential treatments, including therapeutic hypothermia. Our preliminary data showed that a single dose of AZ, injected 15 min after the initiating injury, provided long-term improvements in motor function and reduced brain damage. Our goals are to build on these findings by: i) refining AZ treatment protocols (Aim 1) and ii) evaluating the impact of 3 clinically relevant variables that we are able to assess in this model, on AZ neuroprotective efficacy (Aim 2). Results of these experiments could lay the foundation for clinical trials that test AZ as a neuroprotective therapy in asphyxiated newborns.